Novel amino alcohol dehydrogenase, method for producing said enzyme, and use of said enzyme

ABSTRACT

The present invention provides amino alcohol dehydrogenases which catalyze a reaction to produce keto alcohols, ketones, aldehydes, keto acids from corresponding amino alcohols, amines, and amino acids in the presence of NAD + , and a reaction to produce amino alcohols, amines, amino acids from corresponding keto alcohols, ketones, aldehydes, and keto acids in the presence of NADH and ammonium ions. Also provided is a method for producing the enzymes and uses of the enzyme. The enzymes of the present invention can be obtained from microorganisms belonging to the genera Streptomyces, Pseudomonas, Burkholdenia, or Arthrobacter.

FIELD OF INVENTION

[0001] This invention relates to novel amino alcohol dehydrogenases,methods for preparing the enzymes, and uses of the enzymes.

BACKGROUND OF THE INVENTION

[0002] Amino acid dehydrogenases, amine dehydrogenases,aminotransferases have been known to convert a carbonyl group to anamino group. Amino acid dehydrogenase reductively aminates keto acid toamino acid. Only keto acids and amino acids can be substrates for theenzyme (Experiments of Biochemistry, Vol. 11, ed. by Japan Society ofBiochemistry, Amino acid metabolism and biological amine (I) 193-218, J.Org. Chem., 55, 5567, 1990; Fermentation and Industry 40, 301-311,1982). The inventors examined substrate specificity of commerciallyavailable amino acid dehydrogenases, such as L-alanine dehydrogenase andL-glutamic acid dehydrogenase, and found that they do not have anyenzymatic activity on other amino acid alcohols. In other words, theseNAD(H)-dependent amino acid dehydrogenases only act on very limitedamino acids. Amine alcohols include many useful compounds like syntheticintermediates for pharmaceuticals, such as serinol. Any enzyme that canbe used for synthesizing these amino alcohols has not been reported.

[0003] Amine dehydrogenase uses tryptophane-tryptoquinone (TPQ) or TPQand heme as prosthetic groups, and uses phenazinementasulfate (PMS), anartificial electron carrier, as an electron acceptor. It is independentof NAD(H). This enzyme acts on substrates to produce aldehydes. Thesubstrates include aliphatic 1-amine, such as methylamine, propylamine,n-butylamine, or 1,6-diaminohexane, and some of the enzymes act onarylamine such as 2-phenethylamine or tyramine (Biosci. Biotechnol.Biochem. 62: 469-478, 1998). It does not act, however, on aminoalcohols, amino acids, and aliphatic 2-amines at all.

[0004] Aminotransferase transfers an amino group of an amino acid donorto keto acid, thereby converting the keto acid into amino acid. ω-Aminoacid transaminase or the like, among others, are known to produce anamino compound from ketone, not keto acid (Unexamined Published JapanesePatent Application No. (JP-A) Hei 3-103192, WO97/15682, Appl. Microbiol.Biotechnol. 33, 634-640, 1990, Examined Published Japanese PatentApplication No. (JP-B) Hei 4-11194).

[0005] No alcohol dehydrogenase which converts keto alcohol into aminoalcohol has been reported. The enzymes which converts keto alcohol andketo acid into the corresponding amino alcohol and amino acid, thosewhich converts keto alcohol, ketone, and aldehyde into the correspondingamino alcohol and amine, and those which converts keto alcohol, ketoacid, ketone, and aldehyde into the corresponding amino alcohol, aminoacid, and amine have not been reported. Furthermore, neither methods forproducing such enzymes nor uses of the enzymes have been reported.

SUMMARY OF THE INVENTION

[0006] An objective of the present invention is to provide enzymes thatcan reversibly catalyze the redox reactions described below, productionmethods, and uses of the enzymes.

[0007] As a result of the investigation to achieve the above objective,the present inventors isolated microorganisms producing a noveldehydrogenase that converts keto alcohols into amino alcohols, the onethat concerts keto alcohols and keto acids into the corresponding aminoalcohols and amino acids, the one that converts keto alcohols, ketones,and aldehydes into the corresponding amino alcohols and amines, and theone that converts keto alcohols, keto acids, ketones, and aldehydes intothe corresponding amino alcohols, amino acids, and amines. We purifiedthe enzymes and named them amino alcohol dehydrogenases.

[0008] The present inventors also established a method for producingamino alcohol dehydrogenase, a method for producing amino alcohol fromketo alcohol using the amino alcohol dehydrogenase, a method forproducing amino acid from keto acid, and a method for producing aminefrom ketone or aldehyde.

[0009] Specifically, the present invention relates to an amino alcoholdehydrogenase described below, a method for producing it, and its uses.

[0010] (1) An amino alcohol dehydrogenase that reductively converts ketoalcohol into amino alcohol, and oxidatively converts amino alcohol intoketo alcohol,

[0011] (2) The amino alcohol dehydrogenase of (1), which reductivelyconverts keto acid into amino acid and oxidatively converts amino acidinto keto acid,

[0012] (3) The amino alcohol dehydrogenase of (1) or (2), whichreductively converts ketone or aldehyde into amine and oxidativelyconverts amine into ketone or aldehyde,

[0013] (4) The amino alcohol dehydrogenase of (1), (2) or (3), which isobtainable from a microorganism selected from the group consisting ofthe genera Streptomyces, Pseudomononas, Burkholdenia, and Arthrobacter,

[0014] (5) The amino alcohol dehydrogenase of (4), wherein themicroorganism belonging to the genus Streptomyces is selected from thegroup consisting of the species Streptomyces virginiae, Streptomycesgriseus, Streptomyces avidinii, and Streptomyces pseudovenezulae,

[0015] (6) The amino alcohol dehydrogenase of (4), wherein themicroorganism belonging to the genus Pseudomononas is the speciesPseudomonaonas fluorescens or Pseudomonas marginalis,

[0016] (7) The amino alcohol dehydrogenase of (4), wherein themicroorganism belonging to the genus Burkholdenia is the speciesBurkholdenia cepacia,

[0017] (8) The amino alcohol dehydrogenase of (4), wherein themicroorganism belonging to the genus Arthrobacter is the speciesArthrobacter aurescens,

[0018] (9) An amino alcohol dehydrogenase having the followingphysicochemical properties:

[0019] (a) NAD(H)-dependent;

[0020] (b) a molecular weight of a part of the subunit of about 46,000Da when determined by SDS-polyacrylamide gel electrophoresis, and of thewhole molecule of about 100,000 Da when determined by gel filtration;

[0021] (c) substrate specificity, such that it acts on amino alcohols,amines, amino acids in the presence of NAD⁺ to produce keto alcohols,ketones, aldehydes, and keto acids, and acts on keto alcohols, ketones,aldehydes, and keto acids in the presence of NADH and ammonium ions toproduce amino alcohols, amines, and amino acids;

[0022] (d) thermostability, such that it is relatively stable at 30° C.and inactivated at 40° C. or higher when heated at pH 7.0 for 30 min;

[0023] (e) optimum temperature of about 30 ° C. in reductive aminationat pH 7.0;

[0024] (f) optimum pH of 10.0 in oxidative deamination and of 7.0 inreductive amination; and

[0025] (g) stability, such that its activity is stable in the presenceof glycerol or serinol, or phenylmethylsulfonylfluoride, a proteaseinhibitor,

[0026] (10) A method for producing amino alcohol dehydrogenase, themethod comprising culturing a microorganism, which produces the aminoalcohol dehydrogenase of any one of (1) to (9), and recovering theenzyme from the culture,

[0027] (11) A method for producing amino alcohol, the method comprisingreacting keto alcohol with the amino alcohol dehydrogenase of any one of(1) to (9) in a reaction system, and recovering the corresponding aminoalcohol from the reaction system,

[0028] (12) A method for producing amino acid, the method comprisingreacting keto acid with the amino alcohol dehydrogenase of any one of(2) to (9) in a reaction system, and recovering the corresponding aminoacid from the reaction system,

[0029] (13) A method for producing amine, the method comprising reactingketone and aldehyde with the amino alcohol dehydrogenase of any one of(3) to (9) in a reaction system, and recovering the corresponding aminefrom the reaction system,

[0030] (14) A microorganism producing amino alcohol dehydrogenase of(1), which has the characteristics of the microorganism selected fromthe group consisting of Arthrobacter aurescens B151 identified by adeposit number of FERM P-17137, Burkholdenia cepacia B033 identified bya deposit number of FERM P-17138, Pseudomonas fluorescens B101identified by a deposit number of FERM P-17139, Pseudomonas marginalisB102 identified by a deposit number of FERMP-17140, Streptomyces griseusTPC 33081 identified by a deposit number of FERM P-17141, Streptomycesavidinii A044 identified by a deposit number of FERM P-17142, andStreptomyces pseudovenezulae A161 identified by a deposit number of FERMP-17143.

[0031] The present invention also provides a method for producing ketoalcohol, keto acid, ketone, or aldehyde comprising reacting aminoalcohol, amino acid, or amine with the amino alcohol dehydrogenasedescribed above. In this method as well as the above methods (11) to(13), the microorganism of (14) or its treated product can be used inplace of the amino alcohol dehydrogenase.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Keto alcohol of the present invention can be represented byformula (1):

[0033] wherein R1 and R2 each represents an aliphatic hydrocarbon group,an alicyclic hydrocarbon group, an aryl group, or a heterocyclic group,where these groups are substituted with a hydroxyl group.

[0034] Amino alcohol can be represented by formula (2):

[0035] wherein R1 and R2 are as defined in formula (1).

[0036] Keto acid can be represented by formula (3):

[0037] wherein R3 represents an aliphatic hydrocarbon group, analicyclic hydrocarbon group, an aryl group or a heterocyclic group.

[0038] Amino acid can be represented by formula (4):

[0039] wherein R3 is as defined in formula (3).

[0040] Ketone or aldehyde can be represented by formula (5):

[0041] wherein R4 and R5 each represents a hydrogen atom, an aliphatichydrocarbon group, an alicyclic hydrocarbon group, an aromatichydrocarbon group, or a heterocyclic group, provided that R4 and R5 arenot hydrogen atoms at the same time.

[0042] Amine can be represented by formula (6):

[0043] wherein R4 and R5 are as defined in formula (5).

[0044] An aliphatic hydrocarbon group used herein includes saturated orunsaturated aliphatic hydrocarbon groups. Examples are a straight orbranched alkyl group having 1 to 12 carbon atoms, such as a methylgroup, an ethyl group, a propyl group, an isopropyl group, a butylgroup, an isobutyl group, an s-butyl group, a t-butyl group, a pentylgroup, a hexyl group, an octyl group, a decyl group, etc.; an alkenylgroup having 1 to 12 carbon atoms, such as a vinyl group, an allylgroup, a 1-propenyl group, an isopropenyl group, a 2-butenyl group,etc.; and an alkynyl group having 1 to 12 carbon atoms, such as a2-propynyl group, a 2-butynyl group, etc. An alkyl group having 1 to 5carbon atoms is preferable.

[0045] An alicyclic hydrocarbon group includes saturated or unsaturatedalicyclic hydrocarbon groups. Examples are a cycloalkyl group having 3to 10 carbon atoms, such as a cyclobutyl group, a cyclopentyl group, acyclohexyl group, a cyclooctyl group etc.; and a cycloalkenyl grouphaving 3 to 10 carbon atoms, such as a cyclopentenyl group, acyclohexenyl group, etc.

[0046] An aryl group is, for example, those with 6 to 14 carbon atoms,such as a phenyl group, a naphthyl group, etc.

[0047] A heterocyclic group includes the one containing at least onehetero atom selected from a nitrogen atom, an oxygen atom, and a sulfuratom. A heterocyclic group may be an aromatic heterocyclic group, anon-aromatic heterocyclic group, or a compound heterocyclic group.

[0048] A heterocyclic ring of the above-mentioned heterocyclic groupincludes a nitrogen-containing heterocyclic ring such as pyrroline,pyrrole, piperidine, piperazine, pyridine, pyrimidine, pyridazine,triazole, quinoline, etc.; an oxygen-containing heterocyclic ring suchas tetrahydrofuran, furan, pyran, etc.; a sulfur-containing heterocyclicring such as tetrahydrothiophene, thiophene, etc.; and a heterocyclicring containing at least two hetero atoms selected from a nitrogen atom,an oxygen atom, and a sulfur atom, such as thiazoline, thiazolidine,thiazole, thiazine, morpholine, etc.

[0049] These groups may have substituents, including a halogen atom, ahydroxyl group, an alkyl group (for example, a C₁₋₅ alkyl group such asa methyl group, an ethyl group, a propyl group, an isopropyl group,etc.), an aryl group (for example, a C₆₋₁₄ aryl group such as a phenylgroup, a tolyl group, a chlorophenyl group, a naphthyl group, etc.), anoxo group, an alkoxy group (for example, a C₁₋₅ alkoxy group such as amethoxy group, an ethoxy group, etc.), an aryloxy group (for example, aphenoxy group, etc.), a mercapto group, an alkylthio group (for example,a C₁₋₅ alkylthio group such as a methylthio group, an ethylthio group,etc.), an arylthio group (for example, a C₆₋₁₄ arylthio group such as aphenylthio group, etc.), a carboxyl group, an ester group (for example,a C₁₋₆ alkoxycarbonyl group such as a methoxycarbonyl group, etc.; aC₂₋₁₂ acyloxy group such as an acetoxy group, etc.), an acyl group (forexample, a C₂₋₁₂ acyl group such as an acethyl group, a benzoyl group,etc.), an amino group, a mono- or di-substituted amino group (forexample, a mono- or di- C₁₋₅ alkylamino group such as a methylaminogroup, a dimethylamino group, etc.), a nitro group, etc. The number ofsubstituents is, for example, 1 to 4.

[0050] A preferable keto alcohol in this invention is, for example,hydroxyacetone, dihydroxyacetone, 2-hydroxyacetophenone,4-hydroxy-2-butanone, 5-hydroxy-4-octanone, etc. A preferable keto acidis, for example, pyruvic acid, oxalacetic acid, 2-oxoglutaric acid, etc.A preferable ketone or aldehyde is, for example, acetone, 2-butanone,2-pentanone, 2-hexanone, acetophenone, 4-phenyl-2-butanone,n-butylaldehyde, n-hexylaldehyde, benzaldehyde, etc.

[0051] Microorganisms producing amino alcohol dehydrogenase can beisolated in the following procedures. Microorganisms isolated from thenature, or microorganisms available from depositary institutes arecultured by a standard method. If necessary, a compound that induces theenzyme, such as a substrate, or a compound that enhances the productionof the enzyme, such as metal salts, etc. can be added to the culturemedium. Microbial cells are harvested from the cultured broth, washedwith, for example, buffer, if necessary, disrupted by a mechanicalmethod using alumina, Dyno mill, etc. or treatment with an organicsolvent, such as acetone, etc., to extract the present enzyme. The solidmatters are removed from the extract by filtration or centrifugation toobtain a crude enzyme solution. This solution is added to tris-HClbuffer (pH 8.0-9.0) containing 0.5 mg/ml2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2Htetrazolium chloride(INT), 10 mM serinol, and 1 mM NAD⁺, and the mixture is incubated at 25°C. INT is reduced to red-purple formazan when NAD⁺ is reduced to NADH.Activity of amino alcohol dehydrogenase can be qualitatively orquantitatively measured by this color change.

[0052] A culture medium for the microorganism of this invention containsa carbon source such as glucose, glycerol, etc., which is known to bemetabolized by microorganisms, a nitrogen source such as ammoniumsulfate, ammonium nitrate, etc., inorganic nutrients or metals such asmagnesiumsulfate, iron (II) chloride, cobalt chloride etc. A naturalorganic nitrogen source such as yeast extract, meat extract, etc., canbe added to the medium. Carbon sources adequate for each microorganismcan be applied as an inducer.

[0053] Culture conditions are not particularly limited as long asmicroorganisms can grow. Preferable conditions are, for example, a pHrange of 5 to 10 and a temperature range of 5 to 40° C. High yield canbe obtained by culturing the microorganisms under aerobic conditions ata pH range of 6 to 8, a temperature of 20 to 40° C., for 12 hours to 5days until achieving the maximum activity.

[0054] The enzymatic reaction can be performed by contacting a substratewith microbial cells which are harvested from a liquid medium or a platemedium by a known method. If desired, cells are treated with surfactantsor organic solvents such as toluene to modify cell membranepermeability. The cells can also be immobilized on a supportingmaterials such as carageenan gel, alginate gel, polyacrylamide gel,cellulose, agar, etc. using a known method. The crude enzymes which arepartically purified by the method described below can also be used.These are included in the treated products of the microorganisms usedherein. The microbial cells or their treated products are reacted in atwo-phase system containing a substrate dissolved in an appropriatesolvent such as n-hexane, ethyl acetate, etc. and buffer, etc.Alternatively, a substrate is dissolved in an aqueous organic solventsuch as ethanol, dimethylformamide, etc., and mixed with a suspension ofthe microbial cells, their treated products, or the enzyme.

[0055] Amino alcohol dehydrogenase can be collected from a culturemedium by separating microbial cells and a culture supernatant bycentrifugation or another method. When the enzyme is intracellularlyproduced, the microbial cells are disrupted by, for example, lyticenzyme treatment, ultra-sonication, French press treatment, Dyno milltreatment, etc., to solubilize the enzyme. These treatments are usedalone or in combination.

[0056] The solubilized enzyme can be purified by an appropriatecombination of methods well known in the art. These methods includesalting-out method with, for example, ammonium sulfate, anion exchangechromatography using, for example, diethylaminoethylcellulose, cationexchange chromatography using, for example, carboxymethylcellulose, gelfiltration using, for example, dextran gel, hydrophobic chromatographyusing a hydrophobic resin, and affinity chromatography, etc. An aminoalcohol dehydrogenase preparation with desired purity can thus beobtained.

[0057] When the enzyme is extracellularly produced, culture supernatantis collected by a suitable separation method such as centrifugation andpurified as described above to obtain an amino alcohol dehydrogenasefraction.

[0058] The amino alcohol dehydrogenase of the present invention can beobtained from the culture of microorganisms belonging to, for example,the genera Streptomyces, Pseudomonas, Burkholdenia, or Arthrobacter.

[0059] More specifically, ability to produce amino alcohols wasconfirmed in Streptomyces virginiae IFO 12827 and Streptomyces griseusTPC 33081. IFO 12827 is recited in List of Cultures 10th ed. publishedby Institute of Fermentation, Osaka (IFO) and is available from IFO.

[0060] The present inventors identified the following microorganisms andconfirmed thier ability to produce amino alcohols. These newly isolatedmicroorganisms have been deposited with National Institute of Bioscienceand Human Technology, Agency of Industrial Science and Technology,Ministry of International Trading and Industry of 1-3, Higashi 1-Chome,Tsukuba, Ibaraki 305-0046 Japan) since January12, 1999.

[0061]Arthrobacter aurescens B151, Trust No. FERM P-17137;

[0062]Burkholdenia cepacia B033, deposit No. FERM P-17138;

[0063]Pseudomonas fluorescens B101, deposit No. FERM P-17139;

[0064]Psedomonas marginalis B102, deposit No. FERM P-17140;

[0065]Streptomyces griseus TPC 33081, deposit No. FERM P-17141;

[0066]Streptomyces avidinii A044, deposit No. FERM P-17142; and

[0067]Streptomyces preudovenezulae A161, deposit No. FERM P-17143.

[0068] The present invention provides a method for producing aminoalcohols, amino acids, or amines using the above-mentioned amino alcoholdehydrogenase. Substrates for the enzymatic reaction is keto alcohols,keto acids, ketones, or aldehydes, which provides a basic structure of aproduct compound having amino group(s). The substrate is contacted withthe amino alcohol dehydrogenase of the invention in the presence ofammonium ion as an additional substrate and an electron donor (ahydrogen donor) to perform reduction reaction, thereby producing thecorresponding amino alcohols, amino acids, or amines (aminationreaction). The following reaction formulae illustrate the methods forproducing the above-mentioned compounds according to this invention.

[0069] Compounds used as a substrate in this invention include variousketo alcohols, keto acids, ketones, and aldehydes as described above. Anammonium ion (NH₄ ⁺) that provides an amino group to the basic structureof the substrate is added to the reaction system in the form of anappropriate ammonium salt. The methods of the present invention forproducing amino alcohols, amino aids, or amines can be carried out bycontacting the amino alcohol dehydrogenase of this invention with anelectron donor, NADH, as well as NH₄ ⁺.

[0070] In the reductive amination reaction, 10 to 100 mM of a substrate(keto alcohol, keto acid, ketone, aldehyde), 200 to 300 mM of ammoniumchloride, and 0.2 to 10 mM of NADH can be employed. These substrates andcoenzymes are not necessarily completely dissolved in the reactionmedium. The reaction temperature should be any temperature at which thereaction will proceed, and preferably 10 to 40° C. The pH during thereaction should be from 5 to 8, and preferably at 7. The reductiveconditions can be achieved within the above-mentioned pH range.

[0071] In the oxidative deamination, 10 to 100 mM of a substrate, aminoalcohol, amino acid, or amine, and 0.2 to 10 mM of NAD⁺ can be used. Anytemperature at which the reaction proceeds can be applied, and a rangeof 10 to 40° C. is preferable. A reaction pH should be from 8 to 11, andpreferably 10. The oxidative conditions can be achieved within theabove-mentioned pH range.

[0072] In both reductive amination and oxidative deamination, thesesubstrates and coenzymes are not necessarily completely dissolved in thereaction medium. The substrate can be added at once at the initiation ofthe reaction. Alternatively, it can be added successively orintermittently to the reaction system so that the substrateconcentration becomes too high. The reaction can be allowed to proceedfor about 5 min to about 100 hours. The products can be isolated by aknown method including, for example, extraction, concentration, ionexchange, electric dialysis, crystallization, etc.

[0073] The contact of enzyme, substrate, and coenzyme can be achieved bythe mixing these three in a solution. The reaction solution can be asparingly water-soluble organic solvent such as ethyl acetate, butylacetate, toluene, chloroform, n-hexane, etc., or the two-phase system ofsuch an organic solvent and aqueous medium. The reaction of the presentinvention can be achieved by using immobilized enzymes, membranereactors, etc.

[0074] In the enzyme reaction of the present invention, the reactionconditions become gradually acidic with the consumption of NADH. To keepreductive conditions, a regeneration system of NADH can be combined withthe above-mentioned system. NAD⁺ can be regenerated to NADH by utilizingNAD⁺ reducing ability of microorganisms (glycolysis, Cl compoundmetabolic pathway of methylotroph, etc.). The NAD⁺ reducing ability canbe enhanced by adding glucose, ethanol, or formic acid to the reactionsystem. Alternatively, microorganisms capable of regenerating NADH fromNAD⁺ or their treated products can be added. Such microorganisms are,for example, those producing glucose dehydrogenase, formatedehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organicacid dehydrogenase (such as malate dehydrogenase, etc.), or theirtreated products, partially purified or purified enzymes describedabove. Reactants necessary for NADH regeneration reaction can be addedto the reaction system for producing alcohol of the present invention asthey are, or as their treated products. The reactants can also becontacted with the reaction system through a membrane that enablesexchanging NADH.

[0075] The compound to be added to the reaction system for regeneratingNADH, inlcude, for example, glucose in the case of using glucosedehydrogenase, formic acid in the case of using formate dehydrogenase,ethanol or isopropanol in the case of the using alcohol dehydrogenase,and can be added at a molar ratio to a substrate ketone of 1:20, andpreferably in 1 to 5 times excess amount to a substrate ketone. Theenzymes for regenerating NADH such as glucose dehydrogenase, formatedehydrogenase, or alcohol dehydrogenase can be added in 0.1 to 100times, and preferably 0.5 to 20 times amount of the enzymatic activitycompared with that of the amino alcohol dehydrogenase of the invention.

[0076] Similarly, the reaction system for regenerating NADH to NAD⁺ canalso be combined with the oxidative deamination of the presentinvention. NAD⁺ can be regenerated, for example, using ability tooxidize NADH (NADH oxigenase, etc.) of microorganisms in the presence ofoxygen.

[0077] Amino alcohol dehydrogenase of the present invention has varioususes due to its wide range of substrate specificity. For example, it canbe used for enzymatic synthesis of useful compounds such as serinol.

[0078] Known enzymes converting a carbonyl group to an amino group,including amino acid dehydrogenases, amine dehydrogenases, andaminotransferases can act on limited combinations of keto acid/aminoacid. The amino alcohol dehydrogenases of the present invention areuseful enzymes for solving these problems.

[0079] The present invention is illustrated in detail below withreference to Examples, but not to be construed as being limited thereto.In the following Examples, “%” indicates “w/v%” if not particularlyspecified.

EXAMPLE 1 Isolation of Microorganisms Producing Amino AlcoholDehydrogenase

[0080] A soil sample suspended in saline (0.1 mL) was inoculated onto aplate medium (pH 7.0) containing 0.2% (w/v) serinol, 0.3% KH₂PO₄, 0.1%NaCl, 0.05% MgSO₄.7H₂O, 1.5% agar and cultured at 30° C. under theaerobic condition for 1 to 7 days. The grown colony was inoculated ontothe plate medium containing 0.7% peptone, 0.3% yeast extract, 1.5% agar(pH 7.0) for single colony separation and stored at 4° C. in a slantcontaining 0.2% serinol, 0.3% KH₂PO₄, 0.1% NaCl, 0.05% MgSO₄.7H₂O, 0.1%yeast extract, and 1.5% agar.

[0081] In order to confirm the productivity of amino alcoholdehydrogenase in these stored strains, a crude enzyme solution wasobtained by the following procedures. In the case of actinomyces, 50 mlof a culture medium (pH 7.3) containing 1.5% soluble starch, 0.8%soytone, 0.5% meat extract, 0.3% glucose, 0.2% K₂HPO₄, 0.3% NaCl, 0.03%MgSO₄.7H₂O, 0.01% CaCl₂.2H₂O, 0.1% TM solution was added to anErlenmeyer flask and sterilized. TM solution contains 0.05 g of H₃BO₃,0.01 g of CuSO₄.5H₂O, 0.025 g of KI, 0.1 g FeCl₃.6H₂O, 0.05 gMnCl₂.4H₂O,0.02 g of Na₂MoO₄.5H₂O, 0.05 g of ZnSO₄.7H₂O, 0.01 g ofCoCl₂.6H₂O, and 100 ml of distilled water. Each strain was inoculated tothe sterilized medium and cultured at 30° C. for 48 hours. The microbialcells were then disrupted to obtain a crude enzyme solution.

[0082] In the case of bacteria, 100 ml of a culture media containing0.4% peptone, 0.2% yeast extract, 0.2% 1,3-propanediol, 0.3% KH₂PO₄,0.05% MgSO₄.7H₂O (pH 7.0) were added to a shaking flask, and culturedfor 40 hours in the same manner as for actinomyces. The bacterial cellswere disrupted to obtain a crude enzyme solution.

[0083] This crude enzyme solution was mixed with 0.1M Tris-HCl buffer(pH 9.0) containing 0.5 mg/ml INT, 10 mM serinol, 1 mM NAD⁺ andincubated at 25° C. When NAD⁺ is reduced into NADH, INT is reduced toform red-purple formazan (ε=15000). Based on this reaction, the aminoalcohol dehydrogenase activity in the crude enzyme solution wasspectroscopically determined by measuring the change of absorbance at490 nm. One unit of enzyme was defined as the amount of the enzymeproducing 1 μmol formazan in 1 min under the above condition. Table 1shows serinol dehydrogenase activity of each strain. TABLE 1 Activity(Unit/100 ml Strain of culture medium) Streptomyces virginiae IFO 128270.85 Streptomyces griseus TPC 33081 1.12 Streptomyces avidinii A044 0.06Streptomyces pseudovenezulae A161 0.38 Pseudomonas fluorescens B101 2.72Pseudomonas marginalis B102 3.20 Burkholdenia cepacia B033 1.32Arthrobacter aurescens B151 1.32

EXAMPLE 2 Identification of Microorganisms Producing Amino AlcoholDehydrogenase

[0084] Bacteriological characteristics of microorganisms producing aminoalcohol dehydrogenase isolated from the soil in Example 1 are asfollows.

[0085] Strain A044 has sporogenous hyphae in the hock-like or loop-likeform, or in the form of untightened coil with a few round(Retinaculum-Apertum(RA)). The color of its aerial hyphae is red, andthat of the substrate mycelium is brown. No dispersible pigment isproduced. The production of melanin-like pigment is negative in atyrosine agar medium and is positive in a peptone iron medium.2,6-Diaminopimelic acid, a cell wall component is LL type, and nomycolic acid is detected. Its 16S rDNA has 99.5% or higher homology withStreptomyces avidinii DSM40526T. Strain A044 was thus confirmed tobelong to Streptomyces avidinii species.

[0086] Strain A161 has spiral (Spirae) sporogenous hyphae. The color ofits aerial hypha is gray, and that of the substrate mycelium is brown.No dispersible pigment is produced. The production of melanin-likepigment is negative in both tyrosine agar medium and peptone ironmedium. 2,6-Diaminopimelic acid, a cell wall component is LL type, andno mycoic acid is detected. Its 16S rDNA has 99.5% or higher homologywith Streptomyces pseudovenezulae DSM40212T. Strain A161 was thusconfirmed to belong to Streptomyces pseudovenezulae species.

[0087] Bacteriological characteristics of strain B151 is a gram-positivebacillus (coryneform) with no motility nor sporulation. Both catalasereaction and starch hydrolysis are positive. The type of peptidoglycanof cell walls is A3 α, L-Lys-L-Ala-L-Thr-L-Ala. These characteristicsindicate that strain B151 belongs to the genus Arthrobacter. Homologybetween its 16S rDNA and Arthrobacter aurescens is 98.8% or higher.Strain A161 was thus confirmed to belong to Arthrobacter aurescensspecies.

[0088] Bacteriological characteristics of amino alcoholdehydrogenase-producing strains, B101, B102, and B033 are shown in Table2. Strains B101, B102, and B033 were identified as Preudomonasfluorescens, Pseudomonas marginalis, and Burkholdenia capacia,respectevely. TABLE 2 Characteristics B101 B102 B033 Cell form bacillusbacillus bacillus Cell size 0.5-0.8 to 0.5-0.8 to 0.5-0.8 to 0.8-3.5 μm0.8-3.0 μm 1.5-3.0 μm Motility + + − Flagellum polar flagellum polarflagellum none Gram-stain negative negative negative Spore none nonenone Production of + orange − florescent pigment Catalase + + +Oxidase + + + ADH + + − Nitrate reducing Not tested Not tested − abilityDenitrification + + − ability Homology of 98% 99.8% 99.1% 16SrRNA (P.fluorescens) (P. marginalis) (B. cepacia)

EXAMPLE 3 Cultivation of Microorganisms

[0089]Streptomyces virginiae IFO 12827 was cultured as follows. Oneplatinum loopful of microbial cells from a slant culture was suspendedin 50 ml of a preculture medium (1.0% soluble starch, 0.2% yeastextract, 0.1% meat extract, 0.2% NZ amine, 0.2% malt extract, pH 7.0),inoculated into a sterilized Erlenmeyer flask and shake-cultured at 30°C. for 24 hours under the aerobic condition. Three liters of a mainculture medium (1.5% soluble starch, 0.8% soytone, 0.5% meat extract,0.3% glucose, 0.2% K₂HPO₄, 0.3% NaCl, 0.03% MgSO₄.7H₂O, 0.01%CaCl₂.2H₂O, 0.1% (v/v) TM solution, and 0.1% (w/v) antiform (Antiform A,Sigma), pH 7.3) was added to a 4-liter jar fermentor and sterilized. Theculture broth (50 ml) of the preculture was inoculated therein, andcultured at 30° C. for 48 hours under the aeration conduction at 0.25vvm/400 rpm.

EXAMPLE 4 Cultivation of Microorganisms

[0090]Streptomyces griseus TPC 33081, Streptomyces avidinii strain A044,and Streptomyces pseudovenezulae A161 were cultured in the same manneras in Example 3.

EXAMPLE 5 Cultivation of Microorganisms

[0091] One platinum loopful of Pseudomonas fluorescens B 101 from aslant culture was added in 50 ml of a preculture medium (0.8% peptone,0.2% yeast extract, and 0.3% NaCl (pH 7.0)) and inoculated into asterilized Sakaguchi flask and shake-cultured at 30° C. for 24 hoursunder the aerobic condition. Three liters of a main culture medium (0.4%peptone, 0.2% yeast extract, 0.2% 1,3-propanediol, 0.3% K₂HPO₄, 0.05%MgSO₄.7H₂O, and 0.1% antiform (Antiform A, Sigma) (pH 7.3)) was added toa 4-liter jar fermentor and sterilized. The culture broth of thepreculture was inoculated therein, and cultured at 30° C. for 12 hoursunder the aeration condition at 0.25 vvm/400 rpm.

EXAMPLE 6 Cultivation of Microorganisms

[0092]Pseudomonas marginalis B102, Burkholdenia cepacia B033, andArthrobacter aurescens strain A161 were cultured in the same manner asin Example 5.

EXAMPLE 7 Purification of Enzymes

[0093] Microbial cells were harvested from the liquid culture medium ofStreptomyces virginiae IFO 12827 by centrifugation to obtain about 230 gof wet microbial cells from 4.5 L of the culture broth. The microbialcells were suspended in 92 ml of 20 mM phosphate buffer (KPB, pH 7.0)containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The resultingsuspension was treated with a homogenizer for 3 min and ultra-sonicated(20 kHz, 200 W) for 20 min for disruption. The disrupted products werecentrifuged to obtain supernatant as a crude enzyme solution.polyethyleneimine (0.01% (w/v)) was added to the crude enzyme solution,stirred, and centrifuged to remove precipitates. The supernatant wasdialyzed by ultrafiltration, applied to a Blue-Sepharose column (2.5×24cm) (Pharmacia) to allow the enzyme to pass through-fraction. Thisfraction was collected, concentrated by ultrafiltration and allowed tobe adsorbed by a serine-Sepharose column (2.5×22 cm) equilibrated with10 mM KPB (pH 7.0). The enzyme was eluted by the concentration gradientof KPB containing 0 to 1.2 M NaCl and 20 mM serine. The active fractionwas collected, concentrated and desalted by ultrafiltration, and allowedto be adsorbed by Gigapite column (5.5 cm×20 cm, Seikagaku Corporation).The enzyme was eluted with 5 to 400 mM KPB (pH 7.0). The active fractionwas harvested, concentrated by ultrafiltration, and allowed to beadsorbed by Cellulofine GCL2000sf gel filtration column (1.2×70 cm,Seikagaku Corporation) to elute the enzyme with 10 mM KPB containing 0.1M NaCl. Thus, 8 units of amino alcohol dehydrogenase were obtained at ayield of 10%.

EXAMPLE 8 Purification of Enzyme

[0094] Amino alcohol dehydrogenase was purified from about 100 g ofcultured cells of Pseudomonas fluorescens B101 in the same manner as inExample 7 except for conducting no treatment with a homogenizer. Sixteenunits of amino alcohol dehydrogenase were obtained at a yield of about6%.

EXAMPLE 9 Enzymatic Properties of the Enzyme

[0095] Characteristics of the amino alcohol dehydrogenase derived fromStreptomyces virginiae IFO 12827 obtained in Example 7 were examined.

[0096] 1) Molecular Weight

[0097] The molecular weight of a part of a subunit of the enzymedetermined by SDS-polyacrylamide gel electrophoresis was about 46,000Da, and that of the whole molecule determined by gel filtration wasabout 100,000 Da.

[0098] 2) Coenzyme

[0099] The enzyme is NAD(H)-dependent and does not use NADP(H) as acoenzyme. It does not exhibit any enzymatic activity in the PMS and2,6-dichlorophenolindophenol (DCIP) systems, indicating that it does notuse PMS as an electron acceptor.

[0100] 3) Optimum pH

[0101] The optimum pH for oxidative deamination reaction using serinolas a substrate is 10.0, and that for reductive amination reaction usingdihydroxyacetone is 7.0, as shown in Table 1.

[0102] 4) Optimum Temperature

[0103] The optimum temperature for reductive amination at pH 7.0 isabout 30° C.

[0104] 5) Thermostability

[0105] It is relatively stable at 30° C., and inactivated at 40° C. orhigher when heated for 30 min at pH 7.0.

[0106] 6) Substrate Specificity

[0107] Table 3 show relative activity to various substrates taking theactivity to serinol as 100%. Tables 6 to 8 show relative activity tovarious substrates when taking the activity to dihydroxyacetone as 100%.

[0108] 7) Km Values

[0109] In reductive amination reaction (pH 7.0, phosphate buffer), Km is25 mM when NH₄Cl is substrate, 0.022 mM for NADH, and 2.2 mM fordihydroxyacetone. In oxidative deamination reaction (pH 9.0, Tris-HClbuffer), Km is 0.84 mM for NAD⁺ and 4.0 mM for serinol.

[0110] 8) Stability

[0111] Its activity is stable in the presence of glycerol or serinol, orphenylmethylsulfonylfluoride, a protease inhibitor. TABLE 3 Substrate(Amino alcohols) Relative activity (%) serinol 100 isoleucinol 113L-(−)-methioninol 85 (S)-(+)-leucinol 105 DL-2-amino-1-propanol 2142-amino-2-methyl-1-propanol 115 (+)-2-amino-1-butanol 109DL-2-amino-1-pentanol 103 (S)-(+)-2-amino-3-methyl-1-butanol 43(S)-(−)-2-amino-3-phenyl-1-propanol 120 2-amino-3-hydroxypyridine 1362-aminocyclohexanol 113

[0112] TABLE 4 Substrate (Amino acids) Relative activity (%) L-serine 83L-alanine 160 L-aspartic acid 55 L-glutamic acid 67

[0113] TABLE 5 Substrate (Amines) Relative activity (%) n-butylamine 170n-hexylamine 173 n-octylamine 114 benzylamine 134 (R)-2-aminobutane 1272-aminopentane 252 3-aminopentane 270 (R)-2-aminoheptane 91(R)-1-phenethylamine 127 1-methyl-3-phenylpropylamine 152

[0114] TABLE 6 Substrate (Keto alcohols) Relative activity (%)dihydroxyacetone 100 hydroxyacetone 120 4-hydroxy-2-butanone 1403-hydroxy-2-butanone 89 5-hydroxy-2-pentanone 83 4-hydroxy-3-hexanone 945-hydroxy-4-octanone 83 2-hydroxyacetophenone 186

[0115] TABLE 7 Substrate (Keto acids) Relative activity (%) pyrvinicacid 251 oxalacetic acid 447 2-oxoglutaric acid 107

[0116] TABLE 8 Substrate (Ketones/aldehydes) Relative activity (%)n-butylaldehyde 117 n-hexylaldehyde 100 benzaldehyde 149 acetone 782-butanone 267 2-pentanone 150 2-hexanone 134 acetophenone 1724-phenyl-2-butanone 134

EXAMPLE 10 Enzyme Reaction

[0117] 1) Conversion of Hydroxyacetone into 2-amino-1-propanol

[0118] The amino alcohol dehydrogenase (0.5 unit) obtained in Example 7was added to 2 ml of a mixture containing 5 mM hydroxyacetone, 10 mMNADH, 0.2M NH₄Cl, 0.1 M Tris-HCl buffer (pH 8.0), and the resultingreaction mixture was incubated at 25° C. for 48 hours. The product wasanalyzed by gas chromatography with a FID detector (column, TENAX TA(3.2 mm×1 m); injection and detection temperature, 25° C.; N₂ flow rate,50 ml/min; column temperature, gradient from 150 to 180° C. (5° C./min);retained at 180° C. for 10 min). As a result, the decrease ofhydroxyacetone was detected at the retention time of 2.8 min, and theproduction of 2-amino-1-propanol was detected at the retention time of3.7 min. The retention time of 2-amino-1-propanol was completely thesame as that of the standard compound. These results revealed that theenzyme converted keto alcohol into amino alcohol.

[0119] 2) Conversion of Oxalacetic Acid into Aspartic Acid

[0120] The same reaction system as used in 1) except for usingoxalacetic acid as a substrate in place of hydroxyacetone was incubatedat 25° C. for 48 hours. The produced amino acid was converted into ano-phthalaldehyde (OPA) derivative by OPA-derivatization method. Thederivative was analyzed by high-performance liquid chromatography(column, CAPCELL PAC C18 AG120 (4.6 mm×25 cm, Shiseido); detection, 340nm; column temperature, 45° C.; mobile phase flow rate, 1 ml/ min;concentration gradient elution, a) 10 mM sodium phosphate buffer (pH6.8) to b) acetonitrile:10 mM sodium phosphate buffer (pH 6.8)=2:1). Thepeak of the product, aspartic acid was observed at the retention time of6.5 min, which is completely the same as that of the standard compound.These results indicate that the enzyme converts keto acid into aminoacid.

[0121] 3) Conversion of 1-phenethylamine into Acetophenone

[0122] The amino alcohol dehydrogenase (0.5 unit) was added to a mixtureof 2 mM 1-phenethylamine, 10 mM NAD⁺ and 0.1 M Tris-HCl buffer (pH 9.0),and the resulting reaction solution was incubated at 25° C. for 48hours. The reaction solution was adjusted to pH 10.0 with NaOH andextracted with an equivalent volume of ethyl acetate. The product wasanalyzed by gas chromatography-mass spectrometry (QP-5000GC-MS,Shimadzu, column, DB-1 (0.25 mm×30 m); injection temperature, 180° C.;detection temperature, 250° C.; column temperature, 80° C. retained for5 min, temperature gradient (10° C./min), then retained for 5 min at180° C. The retention time for 1-phenethylamine was 5.42 min and thatfor acetophenone was 5.74 min. The retention time and mass-spectrum ofthe product were completely the same as those for the standard compound.These results indicate that the enzyme converts amine into ketone.

Comparison Example 1 L-alanine Dehydrogenase

[0123] Known NAD(H)-dependent amino acid dehydrogenases reportedly acton only keto acids and amino acids as substrates (Experiments ofBiochemistry, Vol. 11, ed. by Japan Society of Biochemistry, Amino acidmetabolism and biological amine (I) 193-218, J. Org. Chem. 55, 5567,1990; Fermentation and Industry 40, 301-311, 1982). However, it is notreported that the enzymes do not act on amino alcohols at all.Reactivity of a commercially available alanine dehydrogenase to aminoalcohols was examined. Reaction was performed at 25° C. for several minin a reaction solution containing 1 mM NAD⁺, 10 mM of each substrate,0.1 M Tris-HCl buffer (pH 8.0), and 0.02 unit of alanine dehydrogenase(derived from Bacillus stearothermophilius, Seikagaku Corporation). NADHproduced was spectroscopically measured at 340 nm. The enzyme acted onL-alanine but not on L-aspartic acid, L-glutamic acid, serinol,DL-2-amino-1-propanol, 1-phenethylamine, (R)-2-aminobutane, nor2-aminopentane.

Comparison Example 2 L-glutamate Dehydrogenase

[0124] The same experiment as Comparison Example 1 was conducted usingL-glutamate dehydrogenase derived from microorganism (Toyobo), and thatderived from bovine liver (Lifetech Oriental). Both L-glutamatedehydrogenases acted on L-glutamic acid but not on L-alanine, L-asparticacid, serinol, DL-2-amino-1-propanol, 1-phenethylamine,(R)-2-aminobutane, nor 2-aminopentane.

What is claimed is:
 1. An amino alcohol dehydrogenase that reductivelyconverts keto alcohol into amino alcohol, and oxidatively converts aminoalcohol into keto alcohol.
 2. The amino alcohol dehydrogenase of claim1, which reductively converts keto acid into amino acid and oxidativelyconverts amino acid into keto acid.
 3. The amino alcohol dehydrogenaseof claim 1, which reductively converts ketone or aldehyde into amine andoxidatively converts amine into ketone or aldehyde.
 4. The amino alcoholdehydrogenase of claim 2, which reductively converts ketone or aldehydeinto amine and oxidatively converts amine into ketone or aldehyde. 5.The amino alcohol dehydrogenase of claim 1, which is obtainable from amicroorganism selected from the group consisting of the generaStreptomyces, Pseudomononas, Burkholdenia, and Arthrobacter.
 6. Theamino alcohol dehydrogenase of claim 5, wherein the microorganismbelonging to the genus Streptomyces is selected from the groupconsisting of the species Streptomyces virginiae, Streptomyces griseus,Streptomyces avidinii, and Streptomyces pseudovenezulae.
 7. The aminoalcohol dehydrogenase of claim 5, wherein the microorganism belonging tothe genus Pseudomononas is the species Pseudomonaonas fluorescens orPseudomonas marginalis.
 8. The amino alcohol dehydrogenase of claim 5,wherein the microorganism belonging to the genus Burkholdenia is thespecies Burkholdenia cepacia.
 9. The amino alcohol dehydrogenase ofclaim 5, wherein the microorganism belonging to the genus Arthrobacteris the species Arthrobacter aurescens.
 10. An amino alcoholdehydrogenase having the following physicochemical properties: (a)NAD(H)-dependent; (b) a molecular weight of a part of the subunit ofabout 46,000 Da when determined by SDS-polyacrylamide gelelectrophoresis, and of the whole molecule of about 100,000 Da whendetermined by gel filtration; (c) substrate specificity, such that itacts on amino alcohols, amines, amino acids in the presence of NAD⁺ toproduce keto alcohols, ketones, aldehydes, and keto acids, and acts onketo alcohols, ketones, aldehydes, and keto acids in the presence ofNADH and ammonium ions to produce amino alcohols, amines, and aminoacids; (d) thermostability, such that it is relatively stable at 30° C.and inactivated at 40° C. or higher when heated at pH 7.0 for 30 min;(e) optimum temperature of about 30° C. in reductive amination at pH7.0; (f) optimum pH of 10.0 in oxidative deamination and of 7.0 inreductive amination; and (g) stability, such that its activity is stablein the presence of glycerol or serinol, or phenylmethylsulfonylfluoride,a protease inhibitor.
 11. A method for producing amino alcoholdehydrogenase, the method comprising culturing a microorganism, whichproduces the amino alcohol dehydrogenase of claim 1, and recovering theenzyme from the culture.
 12. A method for producing amino alcohol, themethod comprising reacting keto alcohol with the amino alcoholdehydrogenase of claim 1 in a reaction system, and recovering thecorresponding amino alcohol from the reaction system.
 13. A method forproducing amino acid, the method comprising reacting keto acid with theamino alcohol dehydrogenase of claim 2 in a reaction system, andrecovering the corresponding amino acid from the reaction system.
 14. Amethod for producing amine, the method comprising reacting ketone oraldehyde with the amino alcohol dehydrogenase of claim 3 in a reactionsystem, and recovering the corresponding amine from the reaction system.15. A method for producing amine, the method comprising reacting ketoneor aldehyde with the amino alcohol dehydrogenase of claim 4 in areaction system, and recovering the corresponding amine from thereaction system.
 16. A method for producing keto alcohol, the methodcomprising reacting amino alcohol with the amino alcohol dehydrogenaseof claim 1 in a reaction system, and recovering the corresponding ketoalcohol from the reaction system.
 17. A method for producing keto acid,the method comprising reacting amino acid with the amino alcoholdehydrogenase of claim 2 in a reaction system, and recovering thecorresponding keto acid from the reaction system.
 18. A method forproducing ketone or aldehyde, the method comprising reacting amine withthe amino alcohol dehydrogenase of claim 3 in a reaction system, andrecovering the corresponding ketone or aldehyde from the reactionsystem.
 19. A method for producing ketone or aldehyde, the methodcomprising reacting amine with the amino alcohol dehydrogenase of claim4 in a reaction system, and recovering the corresponding ketone oraldehyde from the reaction system.
 20. A microorganism producing aminoalcohol dehydrogenase of claim 1, which has the characteristics of themicroorganism selected from the group consisting of Arthrobacteraurescens B151 identified by a deposit number of FERM P-17137,Burkholdenia cepacia B033 identified by a deposit number of FERMP-17138, Pseudomonas fluorescens B101 identified by a deposit number ofFERM P-17139, Pseudomonas marginalis B102 identified by a deposit numberof FERM P-17140, Streptomyces griseus TPC 33081 identified bya depositnumber of FERM P-17141, Streptomyces avidinii A044 identified by adeposit number of FERM P-17142, and Streptomyces pseudovenezulae A161identified by a deposit number of FERM P-17143.
 21. A method forproducing amino alcohol, the method comprising reacting keto alcoholwith the microorganism of claim 20 or its treated product in a reactionsystem, and recovering the corresponding amino alcohol from the reactionsystem.
 22. A method for producing amino acid, the method comprisingreacting keto acid with the microorganism of claim 20 or its treatedproduct in a reaction system, and recovering the corresponding aminoacid from the reaction system.
 23. A method for producing amine, themethod comprising reacting ketone or aldehyde with the microorganism ofclaim 20 or its treated product in a reaction system, and recovering thecorresponding amine from the reaction system.
 24. A method for producingketo alcohol, the method comprising reacting amino alcohol with themicroorganism of claim 20 or its treated product in a reaction system,and recovering the corresponding keto alcohol from the reaction system.25. A method for producing keto acid, the method comprising reactingamino acid with the microorganism of claim 20 or its treated product ina reaction system, and recovering the corresponding keto acid from thereaction system.
 26. A method for producing ketone or aldehyde, themethod comprising reacting amine with the microorganism of claim 20 orits treated product in a reaction system, and recovering thecorresponding ketone or aldehyde from the reaction system.